CN105543105A - Fungus strain capable of promoting salidroside accumulation of rhodiola crenulata and application of fungus strain - Google Patents

Fungus strain capable of promoting salidroside accumulation of rhodiola crenulata and application of fungus strain Download PDF

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CN105543105A
CN105543105A CN201610011872.7A CN201610011872A CN105543105A CN 105543105 A CN105543105 A CN 105543105A CN 201610011872 A CN201610011872 A CN 201610011872A CN 105543105 A CN105543105 A CN 105543105A
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substratum
radix rhodiolae
mycelia
seedling
accumulation
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CN105543105B (en
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崔晋龙
焦晋
王梦亮
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Shanxi University
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/145—Fungal isolates
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides

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Abstract

The invention discloses a trimmatostroma betulinum fungus strain, and the preservation number is CGMCC No.11417. By co-culturing the fungus strain and a rhodiola crenulata tissue culture seedling or adding a mycelium or a fermentation liquor extract generated through liquid fermentation into a culture medium of the rhodiola crenulata tissue culture seedling, salidroside accumulation of the rhodiola crenulata tissue culture seedling can be promoted. The fungus strain has the important application prospect on rhodiola crenulata resource introduction, promotion of salidroside accumulation of the rhodiola crenulata and the like.

Description

Promote fungal bacterial strain and the application thereof of Radix Rhodiolae accumulation rhodioside
Technical field
The present invention relates to functionally active microorganism, be specifically related to birch powder seat spore fungal bacterial strain, and this fungal bacterial strain and tunning thereof are promoting the application in Radix Rhodiolae tissue cultured seedling accumulation rhodioside.
Background technology
Radix Rhodiolae [Rhodiolacrenulata (Hook.f.etThoms.) S.H.Fu] is the per nnial herb of Rosales Crassulaceae rhodiola.Mainly being distributed in Himalaya area, being grown on height above sea level more than high and cold, anoxic, the intense light irradiation of 3000m, windy mountain area, is the national medicinal plants using more than one thousand years such as local Tibetan.Along with going deep into of research, Radix Rhodiolae medicinal material is widely used in the fields such as medicine, healthcare products, makeup, is the unique Original plant going through the Root of Kirilow Rhodiola medicinal material that version " Chinese Pharmacopoeia " is included.
The chemical composition of Radix Rhodiolae comprises benzene alkylglycosides as styroyl glycoside, tyrosol, rhodioside; Phenylpropanoid Glycosides glycoside (network plug dimension); Phenolic glycoside (kaempferol 7-O--alpha-L-rhamnoside); Flavonoid (glycosides compound of flavonol and formation thereof, as herbaceous stem element 7-O-alpha-L-rhamnoside); Organic acid (as gallic acid), polysaccharide, amino acid, terpenes, alcohols and the inorganic elements such as fats and selenium.Traditional drugs with in, Radix Rhodiolae is mainly used in the treatment of the aspects such as anti-hypoxia, anti-oxidant, antifatigue.Pharmacology activity research shows, rhodioside is one of main active ingredient of Radix Rhodiolae, and the height of its content is as evaluating, and Root of Kirilow Rhodiola kind is good and bad, the important indicator of the Root of Kirilow Rhodiola medicinal material true and false.
At present, the supply of Radix Rhodiolae medicinal material is mainly derived from wild resource, but only has the Determination of Salidroside of perennial rhodiola plant just can reach medicinal standard; Another source be alpine region introduce a fine variety plantation product, even but the Determination of Salidroside accumulation medicinal material of too slow more than 5 years, also lower than wild samples contg; Along with the development of tissue culture technique, people attempt in the wild condition of low altitude area condition Imitating, carry out the extensive tissue culture of Root of Kirilow Rhodiola, but the subject matter faced remain the subject matters such as Determination of Salidroside is on the low side, and accumulating rate is too slow.Therefore, the supply of Root of Kirilow Rhodiola medicinal material is seriously hindered.
The present invention (namely relies at breakthrough conventional art and changes temperature, pH value, illumination, CO 2the condition such as concentration, humidity), utilize fungi and plant symbiosis, the strain that screening obtains is under low altitude area condition, the activity fungal of Root of Kirilow Rhodiola accumulation rhodioside is promoted in the shorter time, this technology has the features such as green is controlled, with low cost, method is novel, and this fungal strain is a kind of biotic induce factor of effective promotion rhodioside accumulation.
Summary of the invention
The invention reside in the biological activity fungi providing a strain can promote Radix Rhodiolae tissue cultured seedling accumulation main active ingredient rhodioside within a short period of time.
A fungal strain ZPRS-R-11 provided by the invention, through being accredited as birch powder seat spore (Trimmatostromabetulinum) bacterium, this fungal strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 6th, 2015, deposit number is CGMCCNo.11417, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The cultural characters of this fungal strain is: cultivate on PDA substratum, and bacterium colony is grey, and mycelia is velveteen shape, central uplift, and mycelia is flourishing, in hair shape edge, without visible secretory product; Bacterium colony back side chocolate, central authorities are dark, and edge is shallow; This bacteria growing speed is partially slow, and the colony diameter growing two weeks is 5cm; Under microscopy conditions, mycelia is in dark-coloured and light color two kinds, and light color mycelia is very thin, and dark-coloured mycelia is sturdy, and diameter, between 1-2 μm, often exists rhizomorph, and mycelia content is high-visible, mycelia without every, tool branch, does not produce spore.
A kind of method promoting the rhodioside Rapid Accumulation organizing training Radix Rhodiolae seedling provided by the invention, it is the bacterial strain activation of CGMCCNo.11417 by deposit number, be inoculated in PDA solid plate substratum, when growth to about plate diameter 2/3 time, with punch tool, bacterium cake is broken into the bacterium cake that diameter is 5 ~ 15mm, get 3 ~ 5 pieces, be evenly inverted on the substratum be put in around Root of Kirilow Rhodiola tissue cultured seedling stem, this substratum is containing 0.3 ~ 1.0mg/LTDZ; 0.3 ~ 1.0mg/LNAA, carries out Dual culture, 15 ~ 25 days, and compared to contrast, the Determination of Salidroside of symbiosis seedling improves greatly.
A kind of method promoting the rhodioside Rapid Accumulation organizing training Radix Rhodiolae seedling provided by the invention, it is the bacterial strain activation of CGMCCNo.11417 by deposit number, be inoculated in PDA solid plate substratum, when growth to about plate diameter 2/3 time, with punch tool, bacterium cake is broken into the bacterium cake that diameter is 5 ~ 15mm, 5 ~ 6 ferfas cakes are inoculated in the triangular flask that the sterilized PDA liquid nutrient medium of 200mL is housed, 25 DEG C ~ 30 DEG C, cultivate 7 ~ 10 days under 150-200rpm condition, by filtered through gauze, obtain mycelia and culture fermentation broth; Mycelia is made mycelia powder under 45 DEG C of conditions, culture fermentation broth is made paste under 45 DEG C of conditions; According to the ratio that weight and volume are 1 ~ 10 ︰ 1000 (g ︰ mL), mycelia and fermented liquid are joined MS substratum respectively (containing 0.3-1.0mg/LTDZ; 0.3-1.0mg/LNAA), inoculation Radix Rhodiolae tissue cultured seedling, cultivate 15 ~ 25 days, compared to contrast, Determination of Salidroside improves greatly.
Compared with prior art, advantage of the present invention and effect:
The present invention adopts deposit number to be birch powder seat spore (Trimmatostromabetulinum) fungi of CGMCCNo.11417 and culture thereof and Root of Kirilow Rhodiola seedling Dual culture, can make Determination of Salidroside Rapid Accumulation.The method breaks through conventional means, adopts microorganism and plant Coculture techniques, and novel, convenient, controlled, development potentiality greatly, is solution Determination of Salidroside good method on the low side.
Embodiment
In embodiment, fungi ZPRS-R-11 is birch powder seat spore (Trimmatostromabetulinum) fungi that deposit number is CGMCCNo.11417.
Embodiment 1
Bacterial strain is activated, be inoculated in PDA solid plate substratum, when growth to about plate diameter 2/3 time, with punch tool, bacterium cake is broken into the bacterium cake that diameter is 5mm, get 3 pieces, be evenly inverted the substratum that is put in around Root of Kirilow Rhodiola tissue cultured seedling stem (containing 0.3mg/LTDZ; 0.3mg/LNAA), carry out Dual culture, after 15 days, the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Embodiment 2
Bacterial strain is activated, be inoculated in PDA solid plate substratum, when growth to about plate diameter 2/3 time, with punch tool, bacterium cake is broken into the bacterium cake that diameter is 15mm, get 5 pieces, be evenly inverted the substratum that is put in around Root of Kirilow Rhodiola tissue cultured seedling stem (containing 1.0mg/LTDZ; 1.0mg/LNAA), carry out Dual culture, after 25 days, the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Embodiment 3
Be that the pure culture biscuits involvng inoculation of 5mm is in the triangular flask that the sterilized PDA liquid nutrient medium of 200mL is housed by 5 pieces of diameters, 25 DEG C, cultivate 7 days under 150rpm condition, by 4 layers of filtered through gauze, obtain mycelium culture thing, dry under 45 DEG C of conditions, grind into powder, is the ratio of 1 ︰ 1000 (g ︰ mL) according to weight and volume, mycelia powder is joined MS substratum (containing 1.0mg/LTDZ; 0.3mg/LNAA), inoculation Radix Rhodiolae tissue cultured seedling, cultivates 15 days, and the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Embodiment 4
Be that the pure culture biscuits involvng inoculation of 15mm is in the triangular flask that the sterilized PDA liquid nutrient medium of 200mL is housed by 6 pieces of diameters, 30 DEG C, cultivate 10 days under 200rpm condition, by 4 layers of filtered through gauze, obtain mycelium culture thing, dry under 45 DEG C of conditions, grind into powder, is the ratio of 10 ︰ 1000 (g ︰ mL) according to weight and volume, mycelia powder is joined MS substratum (containing 3.0mg/LTDZ; 1.0mg/LNAA), inoculation Radix Rhodiolae tissue cultured seedling, cultivates 25 days, and the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Embodiment 5
Be that the pure culture biscuits involvng inoculation of 5mm is in the triangular flask that the sterilized PDA liquid nutrient medium of 200mL is housed by 5 pieces of diameters, 25 DEG C, cultivate 7 days under 150rpm condition, by 4 layers of filtered through gauze, obtain fermented liquid, 45 DEG C of condition rotary evaporations become paste, according to the ratio that weight and volume are 1 ︰ 1000 (g ︰ mL), mycelia powder is joined MS substratum (containing 1.0mg/LTDZ; 0.3mg/LNAA), inoculation Radix Rhodiolae tissue cultured seedling, cultivates 15 days, and the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Embodiment 6
Be that the pure culture biscuits involvng inoculation of 5mm is in the triangular flask that the sterilized PDA liquid nutrient medium of 200mL is housed by 6 pieces of diameters, 30 DEG C, cultivate 10 days under 200rpm condition, by 4 layers of filtered through gauze, obtain fermented liquid, 45 DEG C of condition rotary evaporations become paste, according to the ratio that weight and volume are 10 ︰ 1000 (g ︰ mL), mycelia powder is joined MS substratum (containing 0.3mg/LTDZ; 1.0mg/LNAA), inoculation Radix Rhodiolae tissue cultured seedling, cultivates 25 days, and the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Comparative example 1
Blank: adopt MS substratum (containing 0.5mg/LTDZ; 0.5mg/LNAA), inoculation Radix Rhodiolae tissue cultured seedling, cultivates 20 days, and the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Comparative example 2
PDA liquid nutrient medium 45 DEG C of condition rotary evaporations are become paste by negative control: when not connecing fungi, be the ratio of 5 ︰ 1000 (g ︰ mL), PDA paste joined MS substratum (containing 0.5mg/LTDZ according to weight and volume; 0.5mg/LNAA), inoculation Radix Rhodiolae tissue cultured seedling, cultivates 20 days, and the HPLC method according to " Chinese Pharmacopoeia " P144 page record in 2010 measures Determination of Salidroside.Concrete outcome is in table 1.
Table 1. fungi promotes that Radix Rhodiolae improves rhodioside and tyrosol content
Rhodioside (mg/g)
Embodiment 1 2.216
Embodiment 1 2.309
Embodiment 1 2.782
Embodiment 1 2.752
Embodiment 1 1.963
Embodiment 6 2.351
Comparative example 1 1.450
Comparative example 2 1.487

Claims (5)

1. birch powder seat spore (Trimmatostromabetulinum) fungal bacterial strain, its deposit number is CGMCCNo.11417.
2. the application of fungal bacterial strain in the accumulation promoting Determination of Salidroside in Radix Rhodiolae tissue cultured seedling as claimed in claim 1.
3. one kind promotes the method for the rhodioside Rapid Accumulation organizing training Radix Rhodiolae seedling, step comprises: by fungal bacterial strain activation described in claim 1, be inoculated in PDA solid plate substratum, when growth to about plate diameter 2/3 time, with punch tool, bacterium cake is broken into the bacterium cake that diameter is 5 ~ 15mm, get 3 ~ 5 pieces, be evenly inverted on the substratum be put in around Root of Kirilow Rhodiola tissue cultured seedling stem, this substratum is containing 0.3 ~ 1.0mg/LTDZ; 0.3 ~ 1.0mg/LNAA, carries out Dual culture, 15 ~ 25 days.
4. one kind promotes the method for the rhodioside Rapid Accumulation organizing training Radix Rhodiolae seedling, step comprises: by fungal bacterial strain activation described in claim 1, be inoculated in PDA solid plate substratum, when growth to about plate diameter 2/3 time, with punch tool, bacterium cake is broken into the bacterium cake that diameter is 5 ~ 15mm, 5 ~ 6 ferfas cakes are inoculated in the triangular flask that the sterilized PDA liquid nutrient medium of 200mL is housed, 25 DEG C ~ 30 DEG C, cultivate 7 ~ 10 days under 150-200rpm condition, by filtered through gauze, obtain mycelia and culture fermentation broth; Mycelia is made mycelia powder under 45 DEG C of conditions, is the ratio of 1 ~ 10 ︰ 1000 (g ︰ mL), is joined by mycelia powder in MS substratum according to weight and volume, this substratum is containing 0.3-1.0mg/LTDZ; 0.3-1.0mg/LNAA, inoculation Radix Rhodiolae tissue cultured seedling, cultivates 15 ~ 25 days.
5. one kind promotes the method for the rhodioside Rapid Accumulation organizing training Radix Rhodiolae seedling, step comprises: by fungal bacterial strain activation described in claim 1, be inoculated in PDA solid plate substratum, when growth to about plate diameter 2/3 time, with punch tool, bacterium cake is broken into the bacterium cake that diameter is 5 ~ 15mm, 5 ~ 6 ferfas cakes are inoculated in the triangular flask that the sterilized PDA liquid nutrient medium of 200mL is housed, 25 DEG C ~ 30 DEG C, cultivate 7 ~ 10 days under 150-200rpm condition, by filtered through gauze, obtain mycelia and culture fermentation broth; Culture fermentation broth is made paste under 45 DEG C of conditions; According to the ratio that weight and volume are 1 ~ 10 ︰ 1000 (g ︰ mL), joined respectively by paste in MS substratum, this substratum is containing 0.3-1.0mg/LTDZ, 0.3-1.0mg/LNAA, and inoculation Radix Rhodiolae tissue cultured seedling, cultivates 15 ~ 25 days.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109207375A (en) * 2018-10-09 2019-01-15 山西大学 A kind of method for preserving of plant endogenesis epiphyte strain
CN109797107A (en) * 2019-01-29 2019-05-24 西北大学 A kind of preparation method of hypha,hyphae fine powder
CN114891639A (en) * 2021-11-23 2022-08-12 河北省科学院生物研究所 Microbial strain for improving salidroside content and fermentation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104212725A (en) * 2014-09-15 2014-12-17 山西大学 Method for improving content of salidroside and tyrosol in rhodiola rosea tissue culture seedling
CN104388497A (en) * 2014-12-01 2015-03-04 山西大学 Method for producing salidroside and tyrosol employing phialocephala fortinii

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN104212725A (en) * 2014-09-15 2014-12-17 山西大学 Method for improving content of salidroside and tyrosol in rhodiola rosea tissue culture seedling
CN104388497A (en) * 2014-12-01 2015-03-04 山西大学 Method for producing salidroside and tyrosol employing phialocephala fortinii

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109207375A (en) * 2018-10-09 2019-01-15 山西大学 A kind of method for preserving of plant endogenesis epiphyte strain
CN109797107A (en) * 2019-01-29 2019-05-24 西北大学 A kind of preparation method of hypha,hyphae fine powder
CN109797107B (en) * 2019-01-29 2021-06-04 西北大学 Preparation method of fungus mycelium fine powder
CN114891639A (en) * 2021-11-23 2022-08-12 河北省科学院生物研究所 Microbial strain for improving salidroside content and fermentation method thereof

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